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Biophysik der Molekle
4. Vorlesung Rdler WS 2010
28. Oct. 2010
Protein folding- Afinsen hypothesis- hydrophobic interaction
Gaub/SS 2005 BPM 1.3 2
Protein Unfolding: Sushi Restaurant
When foods with proteins are exposed to heat and certain chemicals (such as vinegar), they turn white.
1. Distinguish salmon roe from imitation salmon
roe by dropping into hot tea.
2. Mackerel is pickled in vinegar for
Gaub/SS 2005 BPM 1.3 3
Nobel Prize for Chemistry in 1972
C. Afinsen 1916-1995
The Thermodynamic Hypothesis (Afinsen 1973)
the native state is thermodynamically stable
=> the sequence alone
determines 3D structure!
loop(usually exposed on surface)
Afinsensmodel protein:ribonuclease A
Ribonuclease kann durch Oxidation (Spaltung der S-S Bindung)denaturiert werden
o) Nofr t iche
Abb. 3.12: Die zwei Zuslnde der Ribonuklease:
links: KomDakt! Funktionsfom
rechts: Oenatudert! Form
b )0eno tu r ie r te
Das Enzym hat 8 s-s Bindungen. Im Prinzip knnten 56 verschiedene Zustnde (Isomere) gebildet werden. Es gibt aber offenbar nur einen Zustand niedrigster Energie.
Folding of RNAse A in the test tube
Incubate proteinin guanidine
100-folddilution of proteininto physiological
Anfinsen, CB (1973) Principles that govern the folding of protein chains.
Science 181, 223-230.
- the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation
Thus: protein folding is a spontaneous process that does not require the assistance of extraneous factors
Folding of proteins in vivo is promoted by chaperones
this bears only on the rate of folding
What drives protein folding?
Minimization of G=E-TS+PV
Minimize the solvation energy.Decrease the conformational entropy.
GFP Fluoreszenz Siehe Biophysik F-Praktikum
Other techniques to probe unfolding
High-resolution techniques (local):
Which forces are dominant in protein folding ?Local vs. non-local interactions
Nonlocal interactions drive collapse transition, whereas local interactions drive helix transitions.
Early model in which protein folding was proposed to be
driven by ion-paired hydrogen bonding among side chains
(Mirsky& Pauling, 1936; Eyring & Stearn, 1939)
disproven by Jacobsen and Linderstrom-Lang
!i=(zie/4"#o)(1/r2) coulomb potential
Sensitive to pH and ion concentrations
pH determines total charge (pI)
Ionic strength determines effective range of interactions
Ion pairs contribute 1-3 kcal/mol (on surface)
Ion pairs generally destabilizing if buried (cost up to 19 kcal/mol/ion to completely bury
Ion pairs contribute ~5-15 kcal/mol per 150 aas
The Kauzmann Hypothesis
hydrophobic interactions determine the thermal stability of the native state
* non-polar solvents denature proteins* unusual temperature dependence: (stability decreases at high AND low temperatures)* protein stability follows same salt dependence as lyotropic (Hofmeister) series
Determination of protein stability.
This can be measured with a variety of tools including, microcalorimetry, spectroscopy, and enzyme function.
The transition can be accomplished with heat or denaturants.
The area under the curve gives $H which agrees with measurements based on the van't Hoff equation
pH Change the ionization state of critical residues
Detergents Bind strongly to the unfolded protein
High concentrations of water soluble organic substances Aliphatic alcohols. These disrupt the water structure
Ionic or polar denaturants including urea and guanidinium
Denaturants: The Hofmeister Series
The ability of an ion to stabilize a protein follows the Hofmeister series
NH4+,Cs+,K+,Na+>Li+>Mg2+>Ca2+>Ba2+ ! ! ! >guanidinium>urea
Thermal stability of RNase A as a
function of salt
This illustrates the effect on protein stability for many commonly used salts.
Potassium phosphate and ammonium sulfate stabilize proteins which accounts for their frequent use in protein purification.
From Voet and Voet second edition
The hydrophobic effect
water forms cluster with coordination number 4
proteins are surrounded bya shell of structured water
= K ,W
0 + RT ln xK ,W
Solubility and partition function
0 = HK
0( ) "T SK0 " SW0( )!
chemical potential,!, and partition coefficient,x of oil molecule in water (w) and oil (K)
" = W
0 = 2.44 + 0.88nC
The entropic change (cost of inducing water order) dominates over the enthalpy change (gain in intermolecular interaction), which is also negative.
" = #4.2 + 0.825nC
in cal/Mol K
63.9 -2.5 -21
84.9 -1.7 -22
105.9 -0.8 -23
! = W
0= 2.44 + 0.88n
Hydrophobic Effect At normal temps the hydrophobic effect is entropic
water molecules form ordered structures around nonpolar compounds
Hydrophobic residues collapse in to exclude water
Additional forces can then stabilize (vdw, h-bond,intrinsic properties)
Hydrophobic effect is dependent on temperature (unstable at high AND low temp).
Thermodynamic considerations Protein stability is composed of two components.
% % $G = $H-T$S
There is a complex temperature dependence for $H and T$S which means that the contribution of the enthalpic and entropic terms changes with temperature.
This temperature dependence arises from the anomalously high change in heat capacity on transferring hydrophobic compounds into water. This is the hall-mark of the hydrophobic effect and arises from the water-ordering.
The heat capacity influences both the temperature dependence of the enthalpy and entropy
It is proportional to the buried non-polar surface area as are all of the thermodynamic parameters.
The large heat capacity is indicative of a well ordered water structure around non-polar molecules in water as is evident from their partial specific volumes when dissolved in water
Temperature dependence of $G
Thermodynamics of transfer of a hydrocarbon from liquid to aqueous solution.
The temperature dependence is the result of different heat capacities of the two phases.
The large changes in $H and T$S compensate so that $G is fairly constant with temperature
Temperature dependence of $H and T$S continued
$H becomes more favorable at lower temperatures, whereas the entropic term becomes less favorable. This is consistent with an increase in the order in the water surrounding the non-polar molecule.
The water-ordering increases the interaction between solvent and solute and thus "enhances" the solubility that would occur in its absence. Even so, the interactions between solute and water eliminate hydrogen bonds within the water that cannot be compensated for by the ordering of the water.
Significantly the van der Waals interactions are greater in the pure water and solute than in the dissolved solute.
It is the loss of hydrogen bonds and van der Waals interactions that is the cause of the hydrophobic interaction.
$H is ~0 at room temperature
Buried hydrophobic surface area The buried hydrophobic surface area for a protein
correlates with the protein stability.
Although it is difficult to predict the overall stability of a pr